Browsing by Subject "Staphylococcus aureus"
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Item Characterization of a novel essential protein Gcp in Staphylococcus aureus(2012-09) Lei, TingThe prevalence of multi-drug resistant Staphylococcus aureus, especially methicillin- and vancomycin-resistant S. aureus has caused serious public health concerns. The limited options for the treatment of multidrug resistant S. aureus infections highlight the urgent need for the identification of novel target and the development of new classes antimicrobial agents. The highly conserved gcp operon is a potential target, as it is essential for all tested bacteria. However, the mechanism behind this essentiality is not clear. In this study, we constructed defined Pspac-regulated gcp or yeaZ expression mutant in wild type MRSA strains and demonstrated the essentiality of Gcp and YeaZ for S. aureus growth in culture. Moreover, we demonstrated that Gcp interacts with YeaZ using both a yeast two-hybrid and a bacterial two-hybrid system, and in vitro pull down assays. To characterize the Gcp-YeaZ interaction, we performed alanine scanning mutagenesis on charged or polarized amino acids of the C-terminal segment of Gcp and revealed that the C-terminal Y317-F322 region was critical for Gcp binding to YeaZ as well as for the essentiality of Gcp for growth. Taken together, our data suggest that the interaction of Gcp and YeaZ may contribute to the essentiality of Gcp for S. aureus survival. In addition, we demonstrated that Gcp and YeaZ play important roles in the regulation of branched-chain amino acids biosynthesis pathway. Specifically, using qPCR and an ilv-promoter luciferase reporter fusion, we found that the depletion of Gcp or YeaZ dramatically increased the transcription of the ilv-leu operon that encodes key enzymes responsible for the biosynthesis of branched-chain amino acids. Moreover, gel shift analyses showed that Gcp lacked a DNA-binding capacity, whereas YeaZ was able to bind the ilv promoter region, indicating that Gcp indirectly, but YeaZ directly control the transcription of ilv-leu operon. Furthermore, we found that Gcp and YeaZ are involved in the biosynthesis of N6-threonylcarbamoyladenosine (t6A). In addition, the depletion of Gcp or YeaZ enhanced bacterial growth in culture medium lacking ILV. To elucidate whether the tight regulation of branched-chain amino acids biosynthesis pathway contributed to the essentiality of Gcp and YeaZ, we created an ilv-leu operon deletion mutant using the defined Pspac-regulated gcp or yeaZ expression strains and found that the deletion of ilv-leu operon had no impact on the requirement of Gcp and YeaZ for bacterial growth. The above data indicate that the essential nature of Gcp and YeaZ is not attributable to their repression of the ilv-leu operon. These new findings provide new insights into the biological function of these essential proteins, Gcp and YeaZ, as well as the regulatory mechanism of branched-chain amino acids biosynthesis.Item Characterization of Staphylococcus aureus at the Human-Swine interface(2016-11) Sun, JisunThe goal of this thesis was to advance understanding of the epidemiology of Staphylococcus aureus in the US swine industry, particularly in relation to potential human health risks from this animal reservoir. The studies were conducted to investigate the prevalence of S.aureus in swine and swine veterinarians in the USA. A total of 720 nasal swabs (20 swabs per herd) were collected from 36 herds and self-nasal samples from 66 swine veterinarians were submitted every month for 18 months. The patterns of S.aureus colonization were determined based on molecular epidemiological methods (MLST and spa typing). Antimicrobial susceptibility testing, enterotoxin genes, immune evasion cluster genes testing were performed to identify their characterizations. Later, a subset of 76 isolates purposely selected was submitted for whole genome sequencing analysis. In swine study, MRSA was not detected in any of the 35 herds except a positive control herd. S.aureus was detected from 76% of pigs sampled. In case of swine veterinarians, the prevalence of S.aureus (avg 65%, monthly range from 58-82%) and MRSA (avg 9%, monthly range from 6-15%) were confirmed suggesting elevated risk of nasal colonization. Similar distribution of genotypes between swine and swine veterinarians were observed. The patterns of phenotypic antibiotic resistance, the absence of enterotoxin genes and IEC genes also showed relatively similar regardless of host species. In addition, WGS data supported that there were unique traits of putative virulence genes and antibiotic resistance genes by each genotype. All the data generated in this dissertation provide a comprehensive assessment of swine associated S.aureus giving a crucial insight into the phenomenon of interspecies dissemination.Item Epidermal Growth Factor Receptor Signaling Mediates the Proinflammatory Effects of Staphylococcus aureus Gamma Toxin on the Mucosa(2016-01-20) Gillman, Aaron N.; Breshears, Laura M.; Torres, Victor J.; Schlievert, Patrick M.; Peterson, Marnie L.; peter377@umn.edu; Peterson, Marnie L.The data are the experimental data used to generate figures for the paper entitled "Epidermal Growth Factor Receptor Signaling Mediates the Proinflammatory Effects of Staphylococcus aureus Gamma Toxin on the Mucosa". The data are being released to comply with PloSONE data availability policy.Item Microbial interactions with epithelial surfaces.(2009-06) Strandberg, Kristi LeeBackground. Staphylococcus aureus is an important gram positive pathogen that causes a multitude of human diseases. Many of these diseases are associated with the production of staphylococcal exotoxins. Superantigens are a family of staphylococcal exotoxins associated with many of these illnesses. Superantigens are defined by their abilities to induce massive cytokine release from host T cells and antigen presenting cells, leading to the development of toxic shock syndrome (TSS). Superantigens have also been associated with atopic dermatitis, an inflammatory skin disease that often coincides with S. aureus colonization of skin lesions. Superantigen production has been shown to induce corticosteroid resistance in human T cells in vitro (corticosteroids are the most common treatment for atopic dermatitis), thus linking superantigens with more severe cases of atopic dermatitis. Past atopic dermatitis studies have focused on select superantigens. To the best of my knowledge, the work presented in this dissertation represents one of the most comprehensive investigation into superantigens associated with S. aureus strains from patients with atopic dermatitis. Superantigens may be also be involved in staphylococcal pulmonary infections. Recently, several community-associated strains of S. aureus have been linked with severe and often fatal pulmonary diseases. I have found that these strains produce superantigens such as toxic shock syndrome toxin-1 (TSST-1), and staphylococcal enterotoxins B (SEB) and C. I hypothesize that superantigens are involved with the severity and fatality associated with these infections (likely by causing TSS). Few treatment options currently exist for preventing staphylococcal toxin production, or neutralizing superantigens in humans. Recent studies have shown exciting evidence that suggests that administration of soluble high affinity T cell receptor subunits (Vβ-TCRs) can neutralize superantigen activity in vitro, and are able to neutralize lethality in a rabbit model of TSS. Further investigations would be useful for determining the full range of toxin-mediated diseases these small peptides could be used to treat. Additional research has demonstrated the potential for using glycerol monolaurate (GML) for inhibiting staphylococcal toxin production in vitro. The ability of GML to inhibit toxin production in vivo has not been studied prior to the work presented in this dissertation. GML has also been shown to inhibit the growth of several vaginal pathogens such as Gardnerella vaginalis, and Candida albicans, without inhibiting the grown of Lactobacillus crispatus, which is a predominant member of the vaginal normal flora. This finding suggests that GML may be useful as a treatment for vaginal infections such as bacterial vaginosis and vulvovaginal candidiasis. Methods. PCR was used to determine the superantigen profiles of S. aureus isolates collected from patients with steroid sensitive atopic dermatitis, steroid resistant atopic dermatitis, and vaginal isolates collected from healthy women. In some cases, superantigen production was quantified using Western immunoblots. Antibiotic sensitivity was determined using disk diffusion assays. SCCmec typing for DNA encoding methicillin-resistance was performed using PCR. To investigate the role of superantigens in severe staphylococcal pulmonary diseases, rabbits were challenged, via the intrabronchial route, with live bacteria (USA200 or USA 400 strains of S. aureus) or purified TSST-1, SEB, or SEC. Some rabbits were hyperimmunized against TSST-1, SEB, or SEC prior to challenge, and some rabbits were administered live bacteria or purified SEB plus soluble high affinity Vβ-TCRs capable of neutralizing SEB superantigenicity. To investigate the effects of GML on staphylococcal exotoxin production and vaginal inflammation, I initiated a double-blind randomized study in which 225 menstruating women were recruited to donate their own used tampons and then wear study tampons (± GML) on their second day of menstruation for 4-6 hours. Tampon samples were screened for the presence of S. aureus, TSST-1, α-hemolysin, and interleukin-8 (IL-8). To investigate further the in vitro and in vivo effects of GML on vaginal microbes, I initiated a small pilot study to determine the effects of GML on vaginal Candida sp, Gardnerella vaginalis, and Lactobacillus sp. In this clinical study, women with chronic vulvovaginal candidiasis and/or bacterial vaginosis were recruited to use an intravaginal gel containing 0%, 0.5%, or 5% GML for 2 days. Swabs of vaginal discharge were collected from the women before and after gel use. Swabs were analyzed for the presence of the microbes of interest, as well as for GML content. Lastly, I investigated severe pulmonary infections caused by USA300 S. aureus strains by challenging rabbits with live bacteria, or purified superantigen. While conducting these studies, I discovered certain USA300 isolates produce a variant form of TSST-1. I used rabbit TSS models, PCR, Southern blots, and superantigenicity assays to begin characterization of the variant form of TSST-1. TSST-1 may cause lethality through mechanisms in addition to superantigenicity. I tested for this lethality by first neutralizing superantigen activity with soluble high affinity Vβ-TCRs, and then administering TSST-1 to rabbits. Results and conclusions. S. aureus isolates collected from steroid-resistant atopic dermatitis patients had significantly different superantigen profiles than other isolates. Strains from patients with steroid-resistant atopic dermatitis carried the genes for a greater number of superantigens, were more likely to produce the three major TSS-associated superantigens (TSST-1, SEB, and SEC), and were also more likely to have uncommon combinations of superantigens, compared to isolates from steroid-sensitive patients and vaginal isolates from healthy women. These findings suggest that S. aureus isolates from patients with steroid-resistant atopic dermatitis are selected for on the basis of greater superantigen production. Although this study focused on isolates collected from steroid-resistant, steroid-sensitive, and from the vaginal mucosa of healthy women, it may also be beneficial to include skin isolates collected from healthy individuals as a control. Both methicillin-sensitive and methicillin-resistant S. aureus (MSSA and MRSA, respectively) colonized patients with steroid-resistant atopic dermatitis. While MSSA and MRSA isolates both had unusual superantigen gene combinations, MSSA isolates had a greater number of superantigen genes. From my studies that focused on the role of superantigens in severe pulmonary diseases, I found that rabbits challenged with viable bacteria developed fatal pulmonary infections. Rabbits previously immunized against purified SEC and then challenged with viable bacteria did not develop fatal disease. Rabbits treated with soluble high affinity Vβ-TCRs for SEB, and then challenged with purified SEB, also did not develop fatal pulmonary disease. In these studies, immunity to superantigens prevents lethality associated with the pulmonary bacterial exposure. Administration of a soluble high-affinity Vβ-TCR to non-immune animals also protected rabbits. Results from in vivo investigations of the effects of tampons containing GML, on both staphylococcal exotoxin production and vaginal inflammation, revealed that lower amounts of S. aureus and exotoxins were detected in study tampons ± GML than the women's own tampons. I also found lower amounts of exotoxins were present in GML+ than GML- study tampons. The chemokine IL-8 was lower in S. aureus- than S. aureus+ tampons and lower in GML+ than GML- study tampons. Collectively, my studies found that tampons containing GML reduce S. aureus exotoxin production. S. aureus increases production of IL-8 in the human vagina, and GML reduces production of this pro-inflammatory chemokine. My findings suggest adding GML to tampons may provide additional safety over other tampons relative to menstrual TSS. My investigations into the effects of GML on additional vaginal pathogens demonstrated that in vitro GML concentrations of ≥100 µg/ml were bactericidal for C. albicans, while concentrations ≥5 µg/ml were bactericidal for G. vaginalis. In human studies, use of either control (0% GML) or GML gels (0.5 and 5% GML) did not significantly alter vaginal pH or levels of Lactobacillus. Use of gels containing GML significantly reduced vaginal levels of Candida. The control gel did not significantly alter Candida levels. Use of both the control and the GML-containing gels significantly lowered the levels of G. vaginalis. These findings suggest that an intravaginal gel containing GML may be useful for treating both vulvovaginal candidiasis and bacterial vaginosis, meanwhile not inhibiting the growth of Lactobacillus. During our studies to investigate the role of superantigens in pulmonary infections caused by USA300 S. aureus isolates, I observed that immunity to staphylococcal enterotoxin-like Q (SEl-Q) offers partial (but not statistically significant) protection from lethal pulmonary infection. Further investigation revealed the presence of a variant form of TSST-1 that contains a large deletion in the tstH gene. This deletion was present in several USA300 clinical isolates. This deletion was predicted to disrupt the ability of variant TSST-1 to stimulate T cells via the Vβ-TCR because variant TSST-1 was predicted to be unable to crosslink the MHC II molecule to the Vβ-TCR. When tested for superantigen activity, variant TSST-1 was found to retain some superantigen activity, and is still highly lethal in rabbits. Additional experiments using soluble high affinity Vβ-TCRs to neutralize full length TSST-1 superantigen activity revealed that full length TSST-1 exposure is still lethal; suggesting that full length TSST-1 can cause lethality through another mechanism in addition to superantigenicity. My findings suggest that lethality due to full length TSST-1 exposure may occur through multiple mechanisms, including superantigenicity, and potentially through direct interaction with the central nervous system.Item A pilot study of the epidemiology of Staphylococcus aureus in multiple site swine production(2013-08) Linhares, Leticia Caldas MonteiroStaphylococcus aureus (S. aureus) is a common colonizer of both humans and pigs (Lowy, 1998; Frana, 2012). The ability of S. aureus to acquire genes that confer resistance to multiple drugs has further elevated its importance to public health (Cuny and Witte, 2008a). In particular, clones of S. aureus that are resistant to methicillin and other beta-lactam antimicrobials (MRSA) are a major clinical problem, and the discovery of MRSA in livestock populations has raised concerns about the potential importance of livestock as reservoirs of MRSA (Voss et al., 2005).However, the importance of pigs in S. aureus transmission to humans and clinical disease is yet to be determined (Cuny and Witte, 2008b). Most recent studies of pigs have focused on MRSA, and there have been no comprehensive studies of the epidemiology of S. aureus (MSSA and MRSA) in pigs. Despite being considered ubiquitous in production animal facilities (Frana, 2012), S. aureus ecology in livestock production farms is poorly documented. Most recent research has used selective enrichment methods to study MRSA in swine populations, rather than generic S. aureus. S. aureus can be isolated from several anatomic sites of pigs, as well as from air, environmental samples and persons having contact with pigs. In fact, isolation of S. aureus in air samples from swine barns suggests this is likely an important route of exposure for people working in livestock facilities (Gibbs et al., 2006; Oppliger et al., 2012). Overall, the limited information on the ecology of S. aureus in the pork production chain limits the ability of the swine industry to understand and communicate the risks to public health in an informed manner.The core rationale for this thesis was that there has been no prior systematic effort to describe the occurrence of S. aureus in swine production systems. The vast majority of studies have focused on MRSA strains using selective culture methods, and/or focused on a limited number of matrices. The objective was therefore to obtain preliminary data on the occurrence of S. aureus in pigs, people, environmental and air samples on pig farms and some insight into the distribution of the organism in the swine farm milieu. Thus, a pilot study of the epidemiology of S. aureus in multiple site swine production was conducted.Item Staphylococcal toxic shock syndrome Toxin-1 interactions with human vaginal epithelial cells and novel therapeutics.(2011-01) Schaefers, Matthew MichaelStaphylococcus aureus is a significant human pathogen that causes a wide range of diseases from skin and soft tissue infections, pneumonia, osteomyelitis, to toxic shock syndrome (TSS). S. aureus initiates infections at skin and mucosal surfaces by producing a multitude of virulence factors, including superantigens (SAgs). SAgs are exotoxins that enhance the ability of S. aureus to cause infection by dysregulation of the host’s immune system. Numerous Staphylococcal SAgs have been identified including the staphylococcal enterotoxins (SEA- SEU) and Toxic Shock Syndrome Toxin-1 (TSST-1). SAgs cause a wide range of diseases such as food-poisoning, atopic dermatitis, and TSS by relatively unknown mechanisms of action on mucosal surfaces. The ability of SAgs to non-specifically cross-link T-cells and antigen presenting cells (APC), which results in a cytokine storm and toxic shock syndrome have been studied extensively. However, interactions of SAgs with mucosal surfaces remain poorly understood. TSST-1, the most common cause of menstrual TSS (mTSS), induces proinflammatory cytokines from vaginal epithelial cells These proinflammatory effects are hypothesized to contribute to the progression of mTSS by disrupting the permeability barrier of the vaginal mucosa directly and by causing a migration of neutrophils, macrophages and lymphocytes to the site of infection. The aims of this thesis were to 1) characterize TSST-1’s mechanism of action on human vaginal epithelial cells (HVEC), leading to the induction of proinflammatory cytokines, including the identification of the TSST-1 HVEC receptor and residues of TSST-1 critical for HVEC interactions, and to 2) evaluate curcumin, an anti-inflammatory compound, as an anti-TSS mucosal therapeutic. HVEC were exposed to TSST-1, and cytokine expression levels were determined by real-time reverse transcription polymerase chain reaction (PCR) and multiplex cytokine assay. IL-6, IL-8, MIP-3α, and TNF- α transcripts were up-regulated (1.5- to 12-fold), with corresponding increases in protein expression. TSST-1 activated an NF-κB luciferase reporter in HVEC, suggesting that NF-κB is a downstream target of TSST-1 signaling. Previous studies have suggested that major histocompatibility complex class II molecules (MHC II) could serve as the epithelial receptors for SAgs, and when activated, induce cytokines. Flow cytometry and Western blotting of HVEC did not detect MHC II molecules. These data suggest that MHC II is not the HVEC surface receptor responsible for induction of cytokines by TSST-1 and another undefined SAg epithelial receptor present on the surface of HVEC is implicated. A dodecapeptide region (TSST-1 amino acids F119 to D130) that is relatively conserved among SAgs has been implicated in SAg penetration of the epithelium. Single amino acid mutations were constructed in TSST-1 amino acids D120 to D130. All mutants maintained superantigenicity similar to wild type toxin. TSST-1 mutants induced IL-8 from HVEC; however, three toxin mutants (S127A, T128A, and D130A) induced lower levels of IL-8 compared to wild type TSST-1. These toxin mutants were administered intravenously to rabbits and all three were 100% lethal. When administered vaginally to rabbits, D130A toxin was nonlethal, while wild type TSST-1 was 100% lethal. Residue D130 may contribute to toxin binding to an epithelial receptor that allows it to penetrate the vaginal mucosa, induce cytokines/chemokines from epithelial cells, and cause TSS. CD40 was explored as a candidate HVEC receptor for TSST-1, based on a previous study which described synergistic activity of CD40 with MHC II for TSST-1 to induce cytokines from monocytes. HVEC (by flow cytometry) and ex vivo human ectocervical tissue (by immunohistochemistry) both expressed CD40. The biological role of CD40 in TSST-1-induced cytokine production was evaluated by reducing CD40 expression in HVEC by stable transfection with small hairpin RNA (shRNA) plasmids by 60% on the protein level. Surprisingly, CD40 shRNA-expressing HVEC produced more IL-8 in response to TSST-1 compared to irrelevant shRNA expressing HVEC. TSST-1 also bound to HVEC expressing shRNA more than HVEC expressing irrelevant shRNA. These data suggest that CD40 is not the HVEC receptor for TSST-1 that is responsible for induction of proinflammatory cytokines. Curcumin, a component of the spice turmeric, is a compound that has been used in traditional medicine therapies for 4,000 years. Curcumin was evaluated as a potential anti-TSST-1 agent by targeting the host mucosal response to TSST-1 and S. aureus. Curcumin inhibited S. aureus exoprotein- or live S. aureus-induced IL-8 production in an ex vivo porcine vaginal tissue model. The importance of TSST-1-induced inflammation of the vaginal mucosa in TSS disease progression was tested by using curcumin to prevent TSS in a rabbit vaginal model. Curcumin co-administrated with TSST-1 intravaginally significantly reduced lethality of TSST-1 by 60% (5 of 8 rabbits survived whereas 0 of 8 rabbits survived in TSST-1 controls, p <0.05). In addition, TNF-α was undetectable from serum or vaginal tissue of curcumin-treated rabbits that survived. These data demonstrate the importance of local inflammation in the progression of TSS, and curcumin as a potential anti-TSS agent. These studies describe the importance of TSST-1 interactions with vaginal epithelial cells in disease progression to TSS. The mechanism of TSST-1 activation of proinflammatory cytokines was determined to be through the NF-κB pathway with TSST-1 amino acid D130 being critical for induction of cytokines from epithelial cells. However, the NF-κB pathway was not induced by TSST-1 binding to MHC II molecules or CD40 suggesting the role of an unidentified epithelial receptor. These studies also determined that curcumin inhibits S. aureus exoprotein-induced cytokine response in ex vivo vaginal porcine tissue; and when administered intravaginally, curcumin partially prevents TSS in a rabbit model of TSS, demonstrating the importance of local inflammation caused by TSST-1 in progression of TSS. These studies were conducted to develop novel therapies for the prevention and treatment of superantigen-mediated diseases.Item Structure and activities of beta toxin: a virulence factor of Staphylococcus aureus(2009-06) Huseby, Medora JeanBeta toxin is a neutral sphingomyelinase secreted by certain strains of Staphylococcus aureus. This virulence factor lyses erythrocytes in order to evade the host immune system as well as to scavenge nutrients. The structure of beta toxin was determined at 2.4 Å resolution using crystals that were merohedrally twinned. This structure is similar to that of the endonuclease HAP1, Escherichia coli endonuclease III, bovine pancreatic DNase I, and the endonuclease domain of TRAS1 from Bombyx mori. Our biological assays demonstrated for the first time that beta toxin kills proliferating human lymphocytes. Structure-directed active site mutations show the biological activities of hemolysis and lymphotoxicity are due to the sphingomyelinase activity of the enzyme. The structures of all bacterial neutral sphingomyelinases solved to date reveal a solvent exposed hydrophobic beta hairpin. We examined the role of this beta hairpin in beta toxin virulence. Altering the length but not the content of the beta hairpin attenuates the biological activities associated with beta toxin. The beta hairpin is an important stabilizing structure. X-ray crystallographic analysis of beta hairpin mutants revealed very minimal structural changes. We show for the first time diacylglycerol bound in the beta toxin truncation (275-280) structure near the beta hairpin region. We also show at 1.75 Å resolution Mg2+ and phosphate bound to the F277A P289A structure. Neutral sphingomyelinases belong to the DNase I super-family of proteins (CATH class 3.60). Beta toxin shares the overall fold of DNase I with an RMSD value 3.3 Å over 220 Cαs. Beta toxin does not function as a DNase and instead precipitates nucleic acid. DNA causes beta toxin to non-specifically cross-link to other proteins. Extracellular DNA is a major structural component of the S. aureus biofilm matrix. Here we demonstrate that beta toxin has a profound effect on forming the matrix on which biofilms grow through the nucleic acid-dependent formation of cross-linked beta toxin monomers as well as other proteins. These links, plus the ability to bind eDNA, enables formation of the underlying nucleoprotein matrix essential to establish a biofilm. The goal of this thesis project is to understand the structural foundations for the role of the virulence factor beta toxin in order to understand the biological mechanism that allows S. aureus to successfully invade, colonize, and attack a host.Item Vaccination and Host-Targeted Interventions for Staphylococcus aureus Exotoxin-Mediated Diseases(2015-05) Gillman, AaronStaphylococcus aureus is a major human pathogen capable of infecting nearly every tissue and causing a wide range of disease including skin and soft tissue infections, pneumonia, osteomyelitis, endocarditis, and toxic shock syndrome. S. aureus produces an array of exotoxins including superantigens and cytolysins, which contribute directly to disease by causing inflammation, immune evasion, and tissue damage. Recent evidence also suggests that superantigens and cytolysins modulate host epithelial cell signaling at mucosal surfaces and contribute directly to disease progression. Antibiotics can eliminate S. aureus, but have no effect on exotoxins already present within tissues. In addition, antibiotic resistance continues to increase in strains of S. aureus. Therefore, I hypothesize that anti-staphylococcal therapies, which target exotoxins or the down-stream host responses to exotoxins, should be explored to treat or prevent S. aureus infections. In this study, I investigated the mechanism of action of the superantigen, Toxic Shock Syndrome Toxin (TSST)-1, and the cytolysins, alpha-toxin and gamma-toxin, at the vaginal mucosal surface. Historically, TSST-1 was determined to promote disease (toxic shock syndrome) by widespread activation of CD4+ T-cells and antigen-presenting cells. However, recently, TSST-1 was determined to have direct proinflammatory effects on vaginal epithelial cells through an epidermal growth factor receptor-dependent pathway. Alpha-toxin’s mechanism of action and synergy with TSST-1 at the vaginal mucosa was characterized using in vitro, ex vivo, and in vivo models. Furthermore, novel actions of gamma-toxin at the vaginal mucosa were identified and the mechanism of action characterized. From this work, I proposed a model of cell-signaling by TSST-1, alpha-toxin, and gamma-toxin at the vaginal mucosa, which demonstrates a conserved proinflammatory and immune-modulating effect by S. aureus exotoxins. Proof of concept was established for two novel therapies for prevention and treatment of S. aureus infections. The tyrosine kinase inhibitor, AG1478, which inhibits the epidermal growth factor receptor signaling pathway, inhibited cytokine production induced by TSST-1, alpha-toxin, and gamma-toxin at the vaginal mucosa and prevented lethal menstrual toxic shock syndrome in rabbits challenged with live S. aureus. In addition, a vaccine targeting multiple exotoxins of S. aureus was highly effective in preventing lethal intrapulmonary challenge with a wide range of clinical strains and for treatment of the lethal effects of exotoxins in rabbits. In summary, these studies describe the mechanism of action of multiple S. aureus exotoxins and provide proof of concept for targeting exotoxins for treatment or prevention of toxin-mediated diseases. These studies demonstrate the importance of local proinflammatory effects during S. aureus infections, which contribute directly to the initiation of clinical disease. The data further highlight the importance of exotoxins during disease and add both insight and complexity to the field of S. aureus pathogenesis.